Allogeneic hematopoietic cell transplantation (HCT) is frequently curative for many otherwise fatal malignant and nonmalignant diseases. However, conventional T cell-replete HCT using pharmacological immunosuppression alone is often complicated by graft versus host disease (GVHD). Mortality attributed to GVHD occurs in 16% of HCT recipients and quality of life is greatly compromised in most patients with moderate or severe chronic GVHD that occurs in 40% of HCT recipients. Moreover, prolonged GVHD precludes the use of T cell immunotherapy to prevent or manage recurrence of leukemia following HCT. T cell depletion is the only current alternative to immunosuppressive drugs and, although effective for preventing GVHD, is complicated by slow immune recovery and high rates of opportunistic infection. The objective of the project is to develop an improved HCT strategy to prevent GVHD, provide for rapid reconstitution of pathogen-specific immunity, and facilitate future immunotherapeutic approaches to reduce relapse in the absence of immunosuppressive drugs. Preclinical studies in murine models by several groups, including by our co-investigator W. Shlomchik, have demonstrated that transplant of purified allogeneic memory T cells (TM) caused less GVHD than did na?ve (TN) or unfractionated T cells. Informed by these studies, we initiated a first-in-human clinical trial to evaluate whether selective depletion of TN from allogeneic PBSC grafts combined with tacrolimus monotherapy could abrogate GVHD and preserve immune reconstitution in recipients of matched related donor (MRD) HCT for acute leukemia. Our preliminary data demonstrates reproducible stem cell engraftment, mild gastrointestinal acute GVHD, a very low incidence of chronic GVHD, rapid reconstitution of pathogen-specific immunity, low levels of relapse and treatment-related mortality and high overall survival. Here, we seek to improve upon these exciting initial results of TN depletion in MRD recipients, and extend this approach to matched unrelated donor (MUD) HCT. The proposed clinical trial will be complemented by detailed correlative and mechanistic laboratory studies, including an in-depth analysis of immune competence and studies to evaluate potential differences in gastrointestinal immune infiltrates and intestinal stem cell damage in recipients of TN-depleted and T cell-replete HCT. The results of the proposed studies have the potential to reduce GVHD and the duration of immunosuppression, which would fundamentally change our approach to allogeneic MRD and MUD HCT, and to provide insight into the pathogenesis of acute GVHD. The specific aims are: 1. To determine whether depletion of TN from PBSC grafts followed by tacrolimus and a short course of methotrexate can reduce GVHD in MRD and MUD HCT recipients. 2. To evaluate reconstitution of pathogen-specific and regulatory T cells, T cell receptor diversity, and immune responses to new antigens delivered by vaccination in TN-depleted and T cell-replete HCT recipients. 3. To evaluate infiltrating T cells and intestinal stem cell damage in recipients of TN-depleted and T cell-replete HCT with GVHD.